Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
The extracellular matrix (ECM) is a complex mixture of collagens, non-collagenous glycoproteins, and proteoglycans that interact to provide a structural scaffold, as well as specific cues for the maintenance, growth and differentiation of cells and tissues. The protein cores of a large number of ECM molecules are composed of different combinations of a finite collection of modules (Engel et al., Development Suppl. 35-42, 1994). The conservation of amino acid sequence of these modules between different ECM proteins and protein families provides us with the opportunity to identify new proteins by database homology searching to help reveal additional modular ECM proteins.
One module present in a number of proteins is the type A-domain, first described in von Willebrand factor (reviewed in Colombatti et al., Matrix 13: 297-306, 1993). Members of the expanding von Willebrand factor type A-domain (VA) protein superfamily participate in a variety of functions including hemostasis, cell adhesion and protein-protein interactions between matrix molecules. ECM components that contain one or more VA domains include collagens types VI (Chu et al., EMBO J. 9: 385-393, 1990; Chu et al., EMBO J. 8: 1939-1946, 1989), VII (Parente et al., Proc. Natl. Acad. Sci. USA 88: 6931-6935, 1991), XII (Yamagata et al., J. Cell Biol. 115: 209-221, 1991), XIV Trueb et al., Eur. J. BioChem. 207: 557, 1992), and XX (Koch et al., J. Biol. Chem. 276: 23120-23126, 2001), matrilins-1, -2, -3, -4 (reviewed in Deak et al., Matrix Biol. 18: 55-64, 1999), cochlin (Robertson et al., Genomics 46: 345-354, 1997), polydom (Gilges et al., BioChem. J. 352: 49-59, 2000) and nine transmembrane α integrin chains (α1, α2, α10, α11, αL, αM, αX, αD and αE) (reviewed in Lee et al., Cell 80: 631-638, 1995), where they are also known as an ‘I’ domain. Non-matrix proteins that contain VA domains include complement system proteins (C2, B) (Mole, J. E., J. Biol. Chem. 259: 3407-3412, 1984), inter-α-trypsin inhibitor (subunits H1-H3) (Chan et al., BioChem. J. 306: 505-512, 1995) α2β subunit of L-type voltage-dependent Ca2+ channel (Ellis et al., Science 241: 1661-1664, 1988) in addition to the archetypal VA domains of von Willebrand factor itself (Sadler et al., Proc. Natl. Acad. Sci. USA 82: 6394-6398).
The crystal structure for several VA domains have been solved including the A1 (Emsley et al., J. Biol. Chem. 273: 10396-10401, 1998) and A3 Bienkowska et al., J. Biol. Chem. 272: 25162-25167, 1997) domains of vWF, and the I domain of integrins αM (Lee et al., 1995, supra), αL (Qu, A. and Leahy, D. J., Proc. Natl. Acad. Sci. USA 92: 10277-10281, 1995) and α2 (Emsley et al., J. Biol. Chem. 272: 28512-28517, 1997). These studies show that the VA module is an independently folding protein unit that attains a classic αβ ‘Rossman’ fold consisting of a parallel β sheet surrounded by amphipathic α helices, and in the majority of VA domains, a metal ion-dependent adhesion site (MIDAS) at the C-terminal end of the β sheet. The MIDAS motif, which consists of five conserved amino acids (DxSxS, T, D), act together with surrounding residues to bind divalent cations and gives I domains of integrins their adhesive and ligand binding properties (Lee et al., 1995, supra). However, not all VA domains contain this motif, for example, the A1 and A3 A-domains of von Willebrand Factor lack some of these conserved amino acids and are not predicted to bind metal ions (Emsley et al., 1998, supra; Bienkowska et al., 1997, supra) and the binding of collagen to the A3 domain is not metal ion dependent (Bienkowska et al., 1997, supra).
VA domains appear to play an important role in protein-protein interactions. In von Willebrand factor, they interact with subendothelial heparans, collagens I, III, (reviewed by Ruggeri, Z. M., J. Clin. Invest. 99: 559-564, 1997) and collagen VI (Denis et al., Arteriosclerosis & Thrombosis 13: 398-406, 1993); in integrins the I domain interacts with several collagens (Tuckwell et al., Eur. J. BioChem. 241: 732-739, 1996); and in collagen VI VA domains interact with heparin Specks et al., EMBO J. 11: 4281-4290, 1992) and collagen IV (Kuo et al., J. Biol. Chem. 272: 26522-26529, 1997). In ECM molecules, the ability of VA domains to interact with other proteins and with each other to promote higher-order structure formation may be crucial in providing a linkage between ECM structural networks. For example, in collagen VI, a specific N-terminal α3(VI) collagen VA domain (N5) is important for the assembly of collagen VI tetramers into functional microfibrils (Fitzgerald et al., J. Biol. Chem. 276: 187-193, 2001) and in matrilin-1, interchain assembly and microfilament formation is promoted by the interaction of the VA domains in adjacent matrilin molecules (Chen et al., Mol. Biol. Cell 10: 2149-2162, 1999).
As described herein, a new member of the VA-domain protein superfamily referred to herein as von Willebrand factor A Related-Protein or WARP has been identified. WARP provides, therefore, a molecular marker of the integrity of the ECM and in particular cartilage. WARP is a novel disulfide-bonded oligomeric ECM glycoprotein that is expressed in cartilage. A genetic sequence encoding WARP is represented herein in itallicized form, i.e., WARP. Both WARP and WARP represent molecular markers of ECM and in particular cartilage integrity. The presence or absence of WARP or altered levels of WARP relative to normal controls is proposed to be indicative of disease conditions such as arthritis or cartilage disease. Furthermore, mutations in WARP are proposed to be genetic indicators of a propensity for a disease condition to occur or provide a diagnostic basis for the presence of a disease condition.